Microbial communities of the gastrointestinal tract profoundly influence the health and immune function of the host. Reciprocally, the host immune system is likely to influence microbiota composition. Traditionally the balance between the host immune response to microbiota and the composition of gut microbiota has been examined in genetically manipulated systems. I propose to use both genetically malleable zebrafish and the evolutionary model organism of the threespine stickleback fish to examine this delicate balance. Studies from the lab of my co-sponsor Karen Guillemin indicate the signaling molecule MyD88 is required for microbiota induced gut immune cell recruitment; I will determine which cell line initiates this recruitment in zebrafish. My preliminary experiments with my co-sponsor William Cresko suggest that the microbiota of evolutionarily distinct populations of marine and freshwater stickleback have different compositions when the fish are raised in the same environment, suggesting that the differences are due to the host genetic background. My initial examination of intestinal innate immune cells in these two populations revealed that the inflammatory response to microbiota is more robust in the marine fish compared to the freshwater populations. I will test the hypothesis that naturally segregated genetic variation in immune responsiveness influences the composition of the gut microbiota. Hypothesis: The strength of the host immune response to gut microbiota shapes the composition of the microbial community. Specific Aims: (1) Determine if MyD88 signaling initiates in gut epithelial or immune cells. (2) Test the hypothesis that the host inflammatory response shapes intestinal microbial communities. (3) Identify genetic components of the immune response that shape microbial communities. Study Design: To explore the mechanism of the immune response to microbiota, I will generate transgenic zebrafish expressing a dominant negative form of MyD88 in intestinal epithelial or immune cells and examine intestinal neutrophil influx and induction of cytokine transcripts in responses to colonization by microbiota. To examine the robustness of the immune response to specific members of the microbiota, I will generate germ free stickleback and compare intestinal neutrophil influx in freshwater or marine fish raised in the presence of simple or complex bacterial communities. To determine if the robustness of inflammation influences microbiota composition, I will manipulate the inflammatory response of these populations and examine the resulting changes in the microbiota using next generation Illumina sequencing of the 16S ribosomal RNA gene. Finally, to identify components of the immune response that shape microbiota communities, I will cross freshwater and marine fish and identify quantitative trait loci that are associated with variation in 1) intestinal microbiota composition and 2) host intestinal gene expression. Candidate host genes associated with differences in microbiota composition and host innate immune responses will be further examined in genetically malleable zebrafish. PUBLIC HEALTH RELEVANCE: Alterations in the bowel microbiota have been linked to numerous diseases including irritable bowel syndrome, inflammatory bowel disease, rheumatoid arthritis, and autism. Reciprocally, both the host innate and adaptive immune responses likely influence the microbiota composition, and yet the host's contribution to this delicate balance is not fully understood. I propose to use two fish models to untangle the immune system contribution to microbiota community membership and the mechanisms of the immune response to microbiota.